The document provides an overview of methods for screening anti-epileptic drugs, including both in vivo and in vitro techniques. It summarizes several common in vivo models used to test drug efficacy, such as the maximal electroshock seizure test and chemically induced seizure models using pentylentetrazol or pilocarpine. It also describes in vitro methods like hippocampal brain slice recordings and receptor binding assays that can be used to study drug mechanisms of action. The document aims to explain techniques for evaluating new compounds and understanding the basic mechanisms of established anti-epileptic medications.

1. Dr.Dheeraj Mulchandani
Department of Pharmacology
Christian Medical College, Ludhiana

2. Overview
 Objectives
 Introduction
 Types of screening
 Different epilepsy/seizure models in vivo
 In vitro methods
 Recent advances
 Summary
 References

3.  To Understand the basic MOA of anti-epileptic drugs.
 Different animal models for epilepsy
 In vivo/ vitro screening techniques
 Different animal models for drug resistant epilepsy

4.  Epilepsy: derived from greek
word “epilepsia” which means
“to seize” or “to hold”
 Disorder characterized by
recurrent unprovoked seizure.
 Seizure: Paroxysmal,
abnormal, excessive, hyper
synchronous discharges from
an aggregate of CNS neurons

5. • Common disorder – 0.5- 0.8% population
• Prevalence of active epilepsy: 5-10 per 1000
• Drug resistant epilepsy seen in 20 - 30% pts

6. Classification
Simple partial
seizures
 Complex partial
seizures
 Partial seizures
with secondary
generalization
 Absence (petit
mal)
 Tonic-clonic
(grand mal)
 Atonic
 Myoclonic
Generalized seizure
Partial seizure

7. • In normally
functioning cortex,
recurrent &
collateral inhibitory
circuits limit
synchronous
discharge amongst
neighbouring
groups of neurons

8. Phenytoin
Valproate
Lamotrigine
CBZ
Barbiturate
Gabapentine
Tiagabine
BZD
AED
Levetiracetam
Lacosamide
Topiramate
Ethosuximide
Valproate
Facilitation of
GABA
mediated Cl-
channel
opening
Inhibition of
excitatory
glutamatergi
c synapse
Prolongation
of Na+ channel
inactivation
Inhibition of
Ca++ mediated T
current

9. Sir Charles
Locock

11. In vivo
models
In vitro
models

13. Electrically induced
seizures
Chemically induced
seizures
Seizures induced by
focal lesions
Genetic models

14.  Maximum Electroshock Seizure
(MES) Test
 Threshold for maximal
electroconvulsions model
 Kindled Rat seizure model
 6 Hz Mouse model

15. • Useful for screening of drugs effective against primary
and secondary GTC
• Animals – rat or mice (8-10)
• Electric stimulation - of cornea or ear with constant
current at 50-60/sec

16. Method (MES)
Evaluation: Anti convulsant property is determined by calculation of ED50
for suppression of tonic hind limb extension.
Observe: Phase of tonic flexion of 1.5 seconds, followed by phase of tonic
extension of about 10seconds, followed by short clonic interval leading to
asphyxial death.
For constant current: 50 mA in mice, 150 mA in rats
Constant Voltage : 250V in mice, 750 V in rats
Electrical stimulation : 50-60 Hz, 0.2 sec corneal or ear electrode

18. …..Maximal Electroshock Seizure(MES) Test
• Evaluation - Suppression of tonic hind limb
extension taken as a measure of efficacy of a drug
against GTC
• e.g: Phenytoin, CBZ, phenobarbitone, primidone
• Disadvantage: Do not give any clue regarding MOA
of the compound.

19.  Aim: To determine the ability of a drug to alter seizure
threshold for tonic limb extension
 Principle: Drugs effective in GTC Seizures ↑threshold
 Animals:
For each stimulus intensity 8-10 mice (male), wt- 18-30
gm

20. Electrical stimulation : 50-60 Hz, 0.2 sec
(constant current or constant voltage)
corneal or ear electrode- 0.2 sec
Observe: Threshold is determined as the current
or voltage inducing hind limb extension in 50%
animals
Calculation : CC50 , CV50
Evaluation: Elevation of threshold by test drug is
taken as a measure of efficacy.
CC50= Current threshold
CV = Voltage threshold

21. Principle:
Process whereby repeated administration of an initial sub-
convulsive electric stimulus results in progressive intensification of
stimulus induced epileptic activity, culminating in generalized
seizures.
Anticonvulsants prevent kindling.
Animals- Female Sprague -Dawley rats; 270-400 gm.

22. Daily
electrical stimulus
strength
400-500 A,
1msec monophasic
Pulses for 1 second
with 50-60 Hz.
Recovery time-
1-2 weeks
Electrode
implanted in
rt. amygdala
(or other
region)
Phase 1 Phase 2 Phase 3

23. During daily stimulation, seizure develops which
evolve through the following steps : RACINE
ö Class 1- Immobility, eye closure, twitching of vibrissae,
stereotypic sniffing.
ö Class 2- Facial clonus and head nodding
ö Class 3- facial clonus, head nodding and U/L forelimb
clonus (C/L to focus).
ö Class 4- Rearing, accompanied b/l forelimb clonus.
ö Class 5- Rearing with loss of balance and falling
accompanied by generalized clonic seizures
– If stimulation continued for few weeks, rats develop spontaneous
epileptic seizures.

24. • Fig. 1. The development of epileptogenesis and emergence of recurrent spontaneous seizures
in the kindling and status epilepticus models. Arrows indicate electrical stimulation in the
kindling model or the administration of chemical agents such as kainate and pilocarpine in
the status epilepticus model. In the kindling model (upper panel), repeated stimulation
triggers progressively stronger seizure responses. If stimulation stops when the animal has
reached the standard stage 5 criterion, spontaneous seizures will not develop. If kindling
stimulations continue, spontaneous seizures typically emerge after the induction of hundreds
of evoked seizures (“over-kindling”).

25.  Test compound : given orally or I/P
 Efficacy determined by
 Seizure latency
 Seizure severity
 Seizure duration

26. • Merits :
a) Efficacy of drug against process of epileptogenesis &
against fully kindled state can be measured
b) Efficacy against Generalized seizures of kindled state
Kindled seizure and kindling process –
Phenobarbitone
Diazepam
Valproic acid
Kindled seizure –
Phenytoin
Carbamazepine

27.  Mice are stimulated by
 Low frequency (6 Hz) electroshock with
 0.2 msec rectangular pulses
 3 sec
 Corneal electrodes
 Result/ end point- Brief clonic convulsion with loss of
righting relexes /stunning behaviour for 10 sec or more
 Use
Screening of Pharmacoresistant epilepsy

29. Chemoconvulsants inducing
generalized seizures after
systemic administration:
PTZ, picrotoxin, penicillin
INH, pilocarpine
NMDA, strychnine
Chemoconvulsants inducing
focal seizures after central
administration:
Penicillin
kainic acid
PTZ

30. • Acts by antagonizing inhibitory GABA transmission
• PTZ is used for screening drugs effective absence
seizures
1) Threshold for clonic seizures after IV infusion of
PTZ
2) S.C injection of PTZ

31. ö Animal – 8-10 mice or rats
ö Dose: 1% PTZ
ö End point : Dose for the production of GTCS with
loss of righting reflex.
ö For petit mal seizures: Ethosuximide & valproate

32. Test group Control
Test drug injected
I.V
No drug
1% PTZ @ 0.3ml/min I.V. 1% PTZ @ 0.3ml/min I.V
Observation -1 hr in
separate plastic cage
Isolated jerk (1st twitch)
GTC with loss of righting reflex
Then maximal tonic clonic seizure
Mean dose of PTZ to
produce seizure-
threshold calculated
Mean dose of PTZ to
produce seizure -
threshold calculated

33. ö Drugs effective in petit mal
epilepsy
ö Ethosuximide
ö Valproate
ö Not effective – phenytoin, CBZ

34. Test group Control
Test drug injected
S.C
No drug
1% PTZ @ 80-100mg s.c 1% PTZ @ 80-100mg s.c
End Point : 1st clonic jerk lasting 5 sec
OR
1st clonic seizure with loss of righting
reflex
SC CD97 determined
30 min
Efficacy of a test drug as an anticonvulsant is measured by its
ED50 for suppression of clonic seizure.

35. • Reduces inhibition by
antagonizing effects of GABA
at its receptor.
• Drug : Penicillin G - 3 lac units/kg I/M.
• Results: Arrested activity, staring, myoclonus,oro-facial
twitching and GTCS, within 1 hr of injection
• This model is useful for screening of drugs in Absence
seizures.
Animal : Cat

37.  Topical or Intracerebral application of metals, chemicals- can lead to simple partial
seizures.
 These models are useful for screening of antiepileptic drugs effective against these
seizures.
1) Cortically implanted metals
Aluminium hydroxide, cobalt powder or tungstic acid injection
2) Aluminium hydroxide gel model – MC used
Animal : monkey
Method : 4% gel injected into neocortex
After 1-2 months spontaneous recurrent simple partial seizures begin which persist
for years.

38. 3) Systemic focal epileptogenesis: model combines features of
focal and generalised epilepsy.
Animal: rats
Method:
• α-particles radiation
given to cerebrum
Destruction of the
blood brain barrier
• After 3-6 months
Inj. bicuculline
2mg/kg i.p
• Seizure is produced with
recurrent EEG spikes and
focal seizures
Last for weeks
Phenytoin, phenobarbitol, valproic
acid

39. Genetic models

40. • Represents true model for epilepsy
• Allow for genetic component of epilepsy
A) Photosensitive baboons (Papio papio):
ピ Intermittent lightening at frequency of 25 flashes/sec
ピ Seizures characterized by eyelid then face, body clonus,
followed by tonic clonic convulsions.
ピ Drugs effective are valproic acid, phenobarbital,
benzodiazepines

41. • 1) Audiogenic suceptible mice- sound induced
seizures:
ピ DBA/2J mice, an inbred strain of house mouse.
ピ Stimulus : Exposed to sudden loud sound 12-16 KHz.
ピ Result : Wild running followed by clonic convulsions,
tonic extension and respiratory arrest (60%) or full
recovery.
ピ Seizures prevented by valproic acid, phenobarbital,
phenytoin.

42. 2) Totterer mice – homozygous (tg/tg) strain
ö Prone to spontaneous epileptic seizures.
ö By 3-4 weeks they develop partial and absence
seizures.
ö Mostly seizures lasts for 15 min or longer.
ö Seizures blocked by ethosuximide,
phenobarbital & diazepam

43. 3) E1 mice
ö E1 mice exhibit Seizures in response to vestibular
stimulation like tossing and spinning.
ö Seizure manifestations – limb and face automatism like
chewing and salivation.
ö Serve as models for complex partial epilepsy with 2˚
generalization.
ö Phenytoin and phenobarbitone are effective.

44. 4) Quaking mice
ö C57BL/6J mutants with myelin defects, tremors, spontaneous
or stimulus induced myoclonic or GTC seizures.
ö Handling induces seizures in Quaking mice.
ö Phenytoin, phenobarbitone, carbamazepine & valproate
ö Useful for assessment of potential new anti convulsant drugs
effective against focal seizures in humans.

45. 1) Genetically epilepsy prone rats (GEPR):
 Seizures can be stimulated by sound, hyperthermia,
chemicals and electricity.
 For anti convulsant drug evaluation – the tonic clonic
component of the seizure is used.
2) Rats with spontaneously occurring Petit Mal
Epilepsy:
• 15-30% Sprague Dawley and wistar rats at 14-18 wks of
age exhibit spontaneous behavioural arrest and myoclonic
twitchings .
• Model for absence seizures

46. ö Seizures can be precipitated by
New environment, bright light,
audiogenic stimulation and
vigrous shaking the cages and
handling techniques.
ö Young animal 7-10 wks - For
Petit mal Epilepsy.

47. ö Syrian golden hamsters
ö Photosensitive epileptic chickens
ö Lethargic mouse
ö Dogs

49. Hippocampal slices
Electrical recording
from isolated brain cells
In vitro assays of
GABAergic compounds
Excitatory Amino Acid
Receptor Binding Assay

50. ö Hippocampus Involved in
generation of complex partial
seizures
ö Useful for studying
neurophysiological
mechanism of epilepsies.
ö Animal: rat , mouse, guinea
pig

51. Rat decapitated
Brain removed
0.5mm slice
using vibrotome
Holding chamber
warm saline(28o),
95% O2 & 5% CO2
Transferred to perspex
chamber & attached to its
bottom
Artificial CSF/ warm saline
Recording from
pyramidal neuron
are done by
micropipette
2 hr.

52. • Illustration of the collection of
hippocampal tissues. With some
practice, the following procedure
takes as little as 15 min from
decapitation of the animal to
placing the cultures in the
incubator. Brains are collected
and prepared (a-c), and
hippocampi are removed (d-f).
Once the tissue chopper is ready
(g), the hippocampi are chopped
(h), transferred to fresh dissection
medium (i), and slices are
separated (j). Ideal slices (k) have
a compact and clearly visible
dentate gyrus granule cell body
layer (D) and pyramidal neuron
cell body layers (CA3 and CA1).
Slices are then placed on tissue
culture inserts (l) for short-term or
long-term culture in a CO2
incubator.

53.  Readings are taken by adding drugs to slice medium
and recording the
 Spontaneous
 Shock evoked repetitive firing of neurons.
Advantage:
•Mechanical stability
•No BBB
•No anaesthetic agent

54.  Isolated brain cells (hippocampus or hypothalamus) are used
for testing action of
 Effect of drug on ion channels in excitable cell membranes
by using Patch clamp techniques
 Use
 Voltage sensitive Ca and K channels
 Membrane response to NT
 Basic mechanism of anti epileptic drugs can be devised

55. 1. GABA receptor binding assay  to evaluate
compounds with GABAergic properties
a. GABAA receptor binding assay
 To examine the GABAA binding sites
 Muscimol (agonist) & SR 95531 (antagonist) are used as
radioligands
b. GABAB receptor binding assay
 Baclofen (agonist)

56. 2) GABA uptake in rat cerebral cortex
 Blocking uptake of GABA by GAT-1 inhibitors tiagabine,
nipecotic acid, guvacine and THPO exhibit anti-convulsant
action

57. 3) TBPS (t- butylbicyclophosphorothionate) binding
assay
 TBPS  convulsant, blocks GABAergic transmission by interacting
with picrotoxin sensitive site
 Inhibition of TBPS binding to channel by test compounds 
used for screening of anticonvulsant drugs

58. • Excessive excitatory neurotransmission implicated in
neuropathogenesis of epilepsy.
1) CPP (3-(2-carboxypirazin-4-yl)-propyl-1-phosphonic acid )
binding assay : Asses affinity of compounds for glutamate
binding site
2) TCP [1-(2-thienyl)cyclohexyl]-piperidine] binding assay :
used for determining the binding affinity of
noncompetitive NMDA receptor antagonist.
3) Glycine binding assay

59. Chemically induced
models
• Pilocarpine induced SE
• Li-Pilocarpine induced
• Li- methomyl induced
Electrically induced
models
• Stimulations in the
hippocampal
prefrontal pathway
Photosensitivity
model
• Photosensitive
baboon- stimulation
by stroboscope

60. University of Utah

64.  Exposure of zebrafish larvae to ginkgotoxin
 Seizure-like swimming behavior that is reversed by commonly
used anti-epileptic drugs
 Provides system for addressing a proposed mechanism of seizure
involving GABA

65.  Neuropeptide Y plays a major role in seizure control.
 Mice lacking the NYP gene experience spontaneous seizures.
 Strategies for NYP gene therapy may develop towards focal
epilepsies targeting Y2 receptor activation.

66.  A molecular genetic tool to treat neurological conditions like
epilepsy.
 Neuronal silencing approach – acting selectively on NpHR (
Halorodopsin, a light sensitive Cloride pump that can
hyperpolarize a neuron when stimulated )
 Optogenetic strategies could be relevant for certain types of focal
seizures.

67. Models used for anti epileptic screening – vivo/vitro
 Most common Animals used: Mice, rat
 also cat, guinea pig, gerbil,
hamster
 MES, PTZ & Kindling are common models
 MES- GTC- Phenytoin, phenobarb
 PTZ- Petit mal epilepsy – ethosuximide, valproate
 Kindling- time consuming-but looks process of
epileptogenesis
 Kindled seizure and kindling process – Phenobarb, Diazepam,
Valprate

68.  Genetic models- best
 Vitro models
 Models of SE – Chemically induced, electrically induced,
photosensitivity models
 Animal models for drug resistant epilepsy
 Recent advances - Zebra fish, NYP gene therapy, optogentics

69. References
• Vogel’s- Drug discovery & evaluation; 3rd edition
• S K Gupta-Drug screening methods. 2nd edition
• Loscher W. Critical review of current animal models of seizure
and epilepsy used in discovery and development of new
antiepileptic drugs. Seizure. 2011;20:359-68
• Magalhães DM, Pereira N, Rombo DM, Beltrão-Cavacas C,
Sebastião AM, Valente CA. Ex vivo model of epilepsy in
organotypic slices—a new tool for drug screening.
Journal of neuroinflammation. 2018 Dec;15(1):203.

70. • Lee et al. Zebrafish larvae exposed to ginkgotoxin exhibit seizurelike
behavior that is relieved by pyridoxal-5-phosphate, GABA and anti-
epileptic drugs. Disease Models & Mechanisms. 2012;5:785-95
Sørensen AT, Kokaia M. Novel approaches to epilepsy
treatment. Epilepsia. 2013 Jan;54(1):1-0.
Löscher W, Brandt C. Prevention or modification of
epileptogenesis after brain insults: experimental
approaches and translational research. Pharmacological
reviews. 2010 Dec 1;62(4):668-700.

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